Reverse transcriptases, enzymes that catalyze RNA-dependent DNA synthesis, have been used as a component of transcription-based amplification systems. These systems amplify RNA and DNA target sequences up to 1 trillion fold. Exemplary systems are disclosed in PCT Patent Application WO 89/01050 to Burg et al.; PCT Patent Application WO 88/10315 to Gingeras et al.; European Patent Application 0 329 822 to Davey and Malek; European Patent Application 0 373 960 to Gingeras et al.; PCT Patent Application WO 91/02814 to Malek and Davey; and European Patent Application 0 408 295 A2 to Kacian and Fultz. Others have also been described or are otherwise commercially available.
Some of the transcription-based amplification methods are exceptionally convenient since the amplification reaction according to these methods is isothermal. Thus, these systems are particularly suited for routine clinical laboratory use in diagnostic tests (i.e., pathogen detection, cancer detection, etc.). Reverse transcriptases are also employed as an initial step in some protocols when the polymerase chain reaction (PCR) is used to amplify an RNA target. See U.S. Pat. No. 5,130,238 to Malek et al.; and Mocharla et al., Gene 99:271–275 (1990). In such “RT-PCR” procedures, the reverse transcriptase is used to make an initial complementary DNA (“cDNA”) copy of the RNA target, which is then amplified by successive rounds of DNA replication.
Reverse transcriptases were once believed to be enzymes unique to the replication of retroviruses (Baltimore, “RNA-dependent DNA Polymerase in Virions of RNA Tumor Viruses,” Nature 226:1209–1211 (1970); Temin and Mizutani, “RNA-Directed DNA Polymerase in Virions of Rous Sarcoma Viruses,” Nature 226:1211–1213 (1970)). Reverse transcriptases are now known to be encoded by a wide range of genetic elements in both eukaryotes and prokaryotes (Varmus, “Reverse Transcription,” Sci. Amer. 257:56–66 (1987); Temin, “Retrons in Bacteria,” Nature 339:254–255 (1989)).
Most commercially available reverse transcriptase, however, are retroviral in origin. The retroviral reverse transcriptases have three enzymatic activities: a RNA-directed DNA polymerase activity, a DNA-directed DNA polymerase activity, and an RNAse H activity (Verma, “The Reverse Transcriptase,” Biochim. Biophys. Acta 473:1–38 (1977)). The latter activity specifically degrades RNA contained in an RNA:DNA duplex. Degradation of the RNA strand of RNA:DNA intermediates by RNAse H is an important component of some transcription-based amplification systems and is to be distinguished from unwanted degradation due to contaminating nucleases, which interferes with amplification. While retroviral-derived reverse transcriptases lacking RNAse H activity have been developed (U.S. Pat. No. 6,063,608 to Kotewicz et al.), it should be noted that retroviral transcriptases are typically characterized by several characteristics which limit their usefulness. These include: the necessity to use an primer that will anneal to the RNA template, the low processivity of the enzymes (i.e., the tendency to dissociate from the RNA before reaching the end), and the inability of the enzymes to transcribe through region of RNA secondary structure.
Eukaryotic genomes in particular are filled with mobile elements, retrotransposons, that use reverse transcriptase for replication. The reverse transcriptases encoded by non-LTR retrotransposons are highly divergent in sequence from the retroviral enzymes and utilize entirely different mechanisms to prime cDNA synthesis.
One of the most abundant classes of reverse transcriptase-encoding elements is the non-LTR retrotransposons (also called LINEs, retroposons and poly A-retrotransposons). Studies of the purified reverse transcriptase from the R2 element of the silkmoth, Bombyx mori, have provided insights into the mechanism of non-LTR retrotransposition (Luan et al., “Reverse Transcription of R2Bm RNA is Primed by a Nick at the Chromosomal Target Site: A Mechanism for non-LTR Retrotransposition,” Cell 72:595–605 (1993)). R2 elements are specialized for insertion into the 28S ribosomal RNA (rRNA) genes found in the nucleoli of eukaryotic cells. The 120 kilodalton protein encoded by R2 has both reverse transcriptase and endonuclease activity. Based on in vitro studies of these two activities, R2 retrotransposition is a coupled DNA cleavage/reverse transcription reaction (Luan and Eickbush, “RNA Template Requirements for Target DNA-Primed Reverse Transcription by the R2 Retrotransposable Element,” Mol. Cell. Biol. 15:3882–3891 (1995); Luan and Eickbush, “Downstream 28S Gene Sequences on the RNA Template Affect the Choice of Primer and the Accuracy of Initiation by the R2 Reverse Transcriptase,” Mol. Cell. Biol. 16:4726–4734 (1996); Mathews et al., “Secondary Structure Model of the RNA Recognized by the Reverse Transcriptase from the R2 Retrotransposable Element,” RNA 3:1–16 (1997); Yang and Eickbush, “RNA-induced Changes in the Activity of the Endonuclease Encoded by the R2 Retrotransposable Element,” Mol. Cell. Biol. 18:3455–3465 (1998); and Yang et al., “Identification of the Endonuclease Domain Encoded by R2 and Other Site-specific, non-Long Terminal Repeat Retrotransposable Elements,” Proc Natl. Acad. Sci. USA 96:7847–7852 (1999)) The 3′ end generated by a first-stand cleavage (nick) of the DNA target site is used as primer for reverse transcription of the RNA template. This utilization of the DNA target to prime cDNA synthesis has been called target-primed reverse transcription (“TPRT”). Removal of the RNA template and synthesis of the second DNA strand does not occur in vitro and is likely to involve the cellular DNA repair and replication machinery. While much has been learned about the TPRT reaction, the activity of any non-LTR element reverse transcriptase has not been characterized in the absence of their DNA target site.
The present invention is directed to overcoming the above-identified limitations of RT reactions performed using previously identified retroviral reverse transcriptases as well as other deficiencies in the art.